Project Summary: The broad, long-term objective of the project is to elucidate mechanisms by which an altered cellular redox environment allows lymphoma cells to evade chemotherapy-induced apoptosis. Steroids are used as a first line therapy for lymphoma and development of resistance to these is a frequent barrier to effective treatment. The hypothesis to be tested is that mitochondria are key to the critical generation of reactive oxygen species during glucocorticoid-induced lymphocyte apoptosis and acquired tolerance to reactive oxygen species is a clinically-relevant mechanism of apoptosis resistance. The specific aims of the proposed research are as follows. 1) To use specific inhibitors of the electron transport chain and p-oxidation of fatty acids, and cells depleted of functional mitochondria, to determine whether the reactive oxygen species which are critical for the signaling phase of glucocorticoid-induced apoptosis derive from mitochondria; and to use a novel fluorescent probe to test for differences in mitochondrial production of superoxide following glucocorticoid treatment of apoptosis-sensitive versus apoptosis-resistant cells. 2) To use immunoblots and 2D gels with proteomic analyses to identify key differences in mitochondrial proteins between glucocorticoid-sensitive and glucocorticoid-resistant lymphoma-derived cell lines; 2D gels will also be immunoblotted to detect significant differences in carbonyl groups that are generated as the result of protein oxidation. 3) To use immunohistochemistry and PCR-based assays to test whether decreased expression of manganese superoxide dismutase occurs in different types of lymphoma and is associated with polymorphisms that alter the transport of this protein into mitochondria or reduce its stability. 4) To use C/EBPp -/- transgenic mice to determine whether loss of this transcription factor in lymphocytes results in an altered cellular redox environment and resistance to apoptosis induced by chemotherapeutic agents used to treat lymphoma. Relevance: The proposed project is aimed at understanding why some lymphomas are not cured by current therapies. The experiments test the idea that treatment-resistant lymphoma cells have changes that make them resistant to oxidative stress and cell death (apoptosis). If this idea is correct, new, more effective anti-lymphoma drugs could be developed to counteract these changes.